PROJECT SUMMARY The Toll-like receptors (TLRs) activated by conserved microbial or endogenous ?danger? molecules trigger intracellular signaling cascades, leading to inflammatory response that is essential for recovery from infection or sterile tissue damage. The excessive TLR response, however, is injurious to the host and can be fatal. The intracellular TLR signaling is initiated by recruitment of TLR adapters to activated TLRs through multiple, homotypic and heterotypic interactions of Toll-Interleukin-1 Receptor homology (TIR) domains present in all TLRs and TLR adapters. Protein interactions mediated by TIR domains, although critical for triggering of signaling, are not well understood due to several intrinsic properties that complicate the analysis of TIR interactions. These complicating factors are (i) the absence of a common TIR-TIR binding motif, (ii) the topological diversity of TIR interface positions, (iii) the ability of each TIR to establish multiple TIR-TIR interactions, (iv) the weakness of any binary TIR-TIR interaction, and (v) the highly cooperative nature of functional TIR-TIR interactions that lead to assembly of TLR signaling complexes. The longstanding goal of this proposal is to advance the molecular understanding of TIR-TIR recognition mechanisms and develop TLR targeted candidate therapeutics that abrogate signaling by blocking the intracellular protein interactions that are consequent to TLR activation. New study will focus on systemic analysis of TIR-TIR interactions that underlie TLR signaling and will decipher the atomic details for several TIR- TIR interactions critical for signal transduction. Specifically, in Aim 1, we will develop novel inhibitors of TLR5, TLR7, TLR8, and TLR9 and evaluate their potency in vivo. Aim 2 is to define the molecular mechanisms of TIR antagonisms by decoy peptides. Studies of Sub-Aim 2.1 will employ the innovative fluorescence imaging approach, which we developed recently, to determine the TIR domains specifically targeted by particular inhibitors in cellular milieu. Sub-Aim 2.2 will employ fluorescence polarization assay (FP) and surface plasmon resonance analysis (SPR) to determine affinity and kinetics of peptide-TIR interactions in controlled environment using recombinant TIR domains. These cellular and in vitro studies will provide complementary and quantitative benchmarks for rapid evaluation of future optimized antagonists. In Sub-Aim 2.3 we will employ X-ray crystallography to decipher the atomic details of TIR-antagonist interactions. Aim 3 is to optimize TIR-derived TLR antagonists using several rational and empirical approaches to increase their target-binding affinity and biological efficacy. The translational aspect of proposed research is the preclinical development of the in vivo potent TLR inhibitors, each with a defined mechanism of action, in several new specificity categories. Importantly, antagonists developed in this project target the intracellular signal transduction machinery. In that they are distinct from candidate therapeutics developed by other groups targeted to block the receptor-ligand interactions and thus our inhibitors may have different biological effects and a wider set of clinical indications.